Confocal imaging of intracellular chloride in living brain slices: Measurement of GABA(A) receptor activity
Confocal imaging of intracellular chloride in living brain slices: Measurement of GABA(A) receptor activity
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DOI：
10.1016/s0165-0270(97)00054-x
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发表时间：
1997-08-22
影响因子：
3
通讯作者：
SchwartzBloom, RD
中科院分区：
文献类型：
--
作者：
Inglefield, JR;SchwartzBloom, RD
我们发展了一种使用紫外激光扫描共聚焦显微镜和荧光氯离子指示剂6-甲氧基-N-乙基喹啉(MEQ)的方法，以成像GABA介导的大鼠急性脑片单个神经元内氯(CI-I)的变化。用MEQ细胞形态(减少)的脑片浸浴后，在海马区、大脑皮层和小脑区的不同形态的神经元内有强烈的荧光。MEQ荧光定位于胞体和近端树突的胞浆室。在不同的细胞外氯离子浓度存在下，使用黑素和三丁基锡离子载体来校准MEQ荧光。神经元MEQ荧光与细胞内氯离子呈负相关，Stern-Volmer常数为16M-1(61 mMCl-猝灭50%)。在灌流液中应用GABA可使MEQ荧光呈浓度依赖性下降(EC50=40µM)，该作用可被氯通道拮抗剂印防己毒素所阻断。用GABA(A)受体调节剂戊巴比妥和安定对海马片进行灌流，可使GABA介导的反应分别增强85%和44%。一项地区性比较发现了更大的GABA。小脑浦肯野细胞和颗粒细胞相对于海马区锥体神经元和新皮质以及海马区中间神经元的反应。从微管向单个海马神经元加压喷射GABA(A)激动剂麝香酚(40µM)，可测量快速反应(1-5 S)，与用浴法得到的结果相比。因此，使用MEQ和UV激光扫描共聚焦显微镜对[Cl-](I)的光学成像为研究人员提供了一种新的方法来研究邻近神经元中的GABA(A)药理学，甚至可能在活体脑片内的胞体与树突中同时研究GABA(A)药理。(C)1997年爱思唯尔爱尔兰科学有限公司。
We have developed a method using UV laser-scanning confocal microscopy and the fluorescent chloride ion indicator, 6-methoxy-N-ethylquinolinium chloride (MEQ), to image GABA-mediated changes in intracellular chloride (Cl-i(-)) in individual neurons of the rat acute brain slice. After bath-loading slices with the cell-permeant form (reduced) of MEQ, there was intense fluorescence within neurons of diverse morphologies in the hippocampus, neocortex and cerebellum. MEQ fluorescence localized to the cytosolic compartment of both the somata and proximal dendrites. MEQ fluorescence was calibrated using the ionophores nigericin and tributyltin in the presence of varying extracellular Cl- concentrations. Neuronal MEQ fluorescence was inversely related to intracellular Cl-, with a Stern-Volmer constant of 16 M-1 (50% quench by 61 mM Cl-). Application of GABA in the perfusate produced a concentration-dependent decrease in MEQ fluorescence (EC50 = 40 mu M) that was blocked in the presence of the Cl- channel antagonist, picrotoxin. Bath perfusion of hippocampal slices with modulators of the GABA(A) receptor, pentobarbital and diazepam, potentiated the GABA-mediated response by 85 and 44%, respectively. A regional comparison identified larger GABA. responses for both cerebellar Purkinje and granule cells relative to pyramidal neurons of the hippocampus and neocortex and to hippocampal interneurons. Pressure ejection of the GABA(A) agonist, muscimol (40 mu M), from a micropipet onto individual hippocampal neurons allowed the measurement of rapid responses (1-5 s), compared to those obtained with bath application. Thus, optical imaging of [Cl-](i) using MEQ and UV-laser-scanning confocal microscopy provides investigators with a new method to study GABA(A) pharmacology in neighboring neurons and perhaps even in the soma versus dendrites, simultaneously, within living brain slices. (C) 1997 Elsevier Science Ireland Ltd.